Molecular toolkit for single-cell oxi-mC analysis

用于单细胞 oxi-mC 分析的分子工具包

• 批准号: 10038441
• 负责人: Yun Huang
• 金额: $ 24.13万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-21 至 2022-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10038441
• 关键词: 3-Dimensional; ATAC-seq; Aberrant DNA Methylation; Adopted; Antibodies; Architecture; Biochemical Reaction; Bioinformatics; Biology; Carbon; Cardiovascular Diseases; Cells; Chemicals; Chromatin; Chromatin Loop; Clinical; Coupling; Cytosine; DNA; DNA Damage; DNA Methylation; DNA Modification Methylases; DNA Modification Process; DNA Probes; Data; Data Set; Development; Dioxygenases; Disease; Epigenetic Process; Exposure to; Family; Gene Expression; Genetic Transcription; Genome; Genomic DNA; Genomic Segment; Genomics; Goals; Heterogeneity; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Individual; Intervention; Knowledge; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Mediating; Methods; Modification; Molecular; Molecular Biology; Molecular Conformation; Monitor; Mus; Nucleotides; Oligonucleotides; Oxides; Pathologic; Play; Positioning Attribute; Preparation; Protein translocation; Reaction; Research; Research Personnel; Resolution; Role; Sampling; Side; Switch Genes; Techniques; Technology; Testing; Tissues; Treatment Protocols; Variant; analysis pipeline; base; biological systems; biomaterial compatibility; bisulfite; chemical reaction; comparison group; computerized data processing; cost; demethylation; disease diagnosis; embryonic stem cell; epigenome; epigenome editing; functional genomics; genome integrity; genome-wide; human disease; innovation; innovative technologies; methyl group; oxidation; response; technology development; technology research and development; tool

Project Summary / Abstract DNA methylation homeostasis is primarily maintained by coordinated actions of DNA methyltransferases (DNMTs) and the Ten-eleven Translocation (TET) dioxygenase. While DNMTs catalyze the addition of a methyl group to the carbon-5 position of cytosine to generate 5- methylcytosine (5mC), TET proteins mediate the further oxidation of 5mC to successively yield 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These DNA methylation oxidation products, collectively termed as “oxi-mC”, not only serve as critical intermediates during active DNA demethylation, but also act as important epigenetic marks to regulate gene transcription, chromatin accessibility and 3D chromatin organization. With advanced sequencing technology, the genomic distribution and function of individual oxi-mC species are just beginning to be appreciated. However, our current knowledge on oxi-mC is mostly based on sequencing results from bulk cells and tissues. A major current challenge is to efficiently probe single cell oxi-mC at single-base resolution, and to unambiguously assign their correlations with 3D chromatin features. The existing single cell 5hmC and 5fC mapping technologies adopt totally different strategies, including antibody- based enrichment, chemical/enzymatic reactions, and/or bisulfite treatment. The variation in sample treatment protocols adds tremendous complexity for library preparation and introduces significant barriers for data processing and cross-group comparisons. By leveraging complementary expertise in epigenetics, chemical biology and bioinformatics, the PI has assembled a strong team of investigators to tackle this challenge. The goal of this exploratory technology development proposal is to develop an innovative and widely applicable molecular toolset, which will enable paralleled high-resolution mapping and analysis of individual oxi-mC heterogeneity and chromatin architectures in single cells derived from various biological systems. Given the importance of chromatin accessible regions and long-range chromatin loops in governing gene expression, we will focus on unraveling the functional coupling of oxi-mC with 3D chromatin organization in these functional genomic regions. The successful execution of this project will provide a streamlined pipeline to profile oxi-mC at single-base resolution and permit simple side-by-side comparison of 5hmC, 5fC and 5caC dynamics. This toolkit can be widely used to monitor oxi-mC dynamics in normal development and various pathological conditions associated with aberrant DNA methylation, such as cardiovascular disease, immunoinflammatory disorders and cancer.

项目总结/摘要 DNA甲基化稳态主要通过DNA的协调作用来维持。 甲基转移酶（DNMT）和10 - 11易位（泰特）双加氧酶。而DNMT 催化甲基加成至胞嘧啶的碳-5位以生成5-甲基胞嘧啶， 甲基胞嘧啶（5 mC），泰特蛋白介导5 mC的进一步氧化，以相继产生5-甲基胞嘧啶（5 mC）。 羟甲基胞嘧啶（5 hmC）、5-甲酰基胞嘧啶（5 fC）和5-羧基胞嘧啶（5caC）。这些DNA 甲基化氧化产物，统称为“oxi-mC”，不仅作为关键的中间体， 在活跃的DNA去甲基化过程中，也作为重要的表观遗传标记调节基因 转录、染色质可及性和3D染色质组织。先进的测序技术 技术，个别oxi-mC物种的基因组分布和功能才刚刚开始 赞赏.然而，我们目前对oxi-mC的了解主要是基于来自 散装细胞和组织。目前的一个主要挑战是有效地探测单细胞oxi-mC在单碱基 分辨率，并明确分配其与3D染色质特征的相关性。现有 单细胞5 hmC和5 fC定位技术采用完全不同的策略，包括抗体- 基于富集、化学/酶促反应和/或亚硫酸氢盐处理。样本变异 处理方案增加了文库制备的巨大复杂性， 数据处理和跨组比较的障碍。通过利用互补的专业知识， 表观遗传学，化学生物学和生物信息学，PI已经组建了一支强大的研究团队 来应对这一挑战。这项探索性技术开发提案的目标是开发一种 创新和广泛适用分子工具集，这将使高分辨率绘图成为可能 和分析单个oxi-mC的异质性和染色质结构， 各种生物系统。考虑到染色质可及区域和长距离染色体的重要性， 染色质环在控制基因表达，我们将集中在解开的功能耦合 oxi-mC在这些功能基因组区域中具有3D染色质组织。成功执行 该项目的一部分将提供一个简化的管道，以单碱基分辨率分析oxi-mC，并允许 5 hmC、5 fC和5caC动力学的简单并排比较。该工具包可广泛用于 监测正常发育和与之相关的各种病理状况中的oxi-mC动力学 异常的DNA甲基化，如心血管疾病、免疫炎症性疾病和癌症。